PROJECT SUMMARY This renewal application of our long-term effort to understand how IKK-dependent NF-?B signaling controls inflammation and immunity is focused on positive and negative regulation of the NLRP3 inflammasome by NF-?B and mitochondrial (mt) metabolism. Our effort will be placed on complete elucidation of a novel signaling mechanism, identified in our laboratory, through which engagement of Toll-like receptors (TLR) renders macrophages (M?) responsive to stress, damage signals and microparticles that trigger NLRP3 inflammasome activation and induce IL-1? and IL-18 production. Persistent NLRP3 inflammasome activation is involved in several neurodegenerative, metabolic and inflammatory diseases, e.g. Alzheimer?s disease, type II diabetes and osteoarthritis (OA), but its poor understanding has prevented development of novel NLRP3-specific anti- inflammatory drugs. Furthermore, previous attempts to alleviate inflammation by targeting IKK-dependent NF- ?B signaling have failed due to enhanced NLRP3 inflammasome activation. By studying how NF-?B negatively regulates the NLRP3 inflammasome, we identified a critical role for mitochondria in control of inflammasome activity. Whereas, NF-?B- and p62-dependent mitophagy terminates NLRP3 inflammasome activation in stimulated M?, TLR4 or TLR3 engagement triggers mtDNA replication via a novel, pathway based on activation of IRF-1 and induction of the nucleotide kinase CMPK2. This pathway is essential for production of oxidized (Ox) mtDNA in TLR-activated M? that were exposed to diverse NLRP3 inflammasome activators, e.g. ATP, nigericin, alum and DOTAP liposomes. Our results suggest that Ox-mtDNA is the ultimate NLRP3 ligand responsible for inflammasome assembly and activation. We will continue to study this pathway and investigate the suitability of its targeting for treatment of currently incurable inflammatory diseases, such as OA. Accordingly, we will determine whether CMPK2 knockout and knockin mice exhibit defective NLRP3 inflammasome activation and are therefore resistant to hydroxyapatite-induced joint inflammation. We will also determine how CMPK2- dependent mtDNA replication supports Ox-mtDNA production and examine whether the latter binds NLRP3 directly, map the binding site and conduct biochemical and structural studies to determine how Ox-mtDNA binding induces the association of NLRP3 with the inflammasome scaffold protein ASC. We will investigate how other mt signals and metabolites, reactive oxygen species (ROS) and itaconic acid (IA), modulate synthesis of pro-IL-1?, pro-IL-18 and other cytokines. These studies will focus on the role of the oxidant-responsive transcription factor NRF2 in cytokine gene expression and will also explore how mtROS and IA affect NF-?B activity and its crosstalk with NRF2. These studies will expand our basic understanding of the fundamental mechanisms that control inflammation and will lay the foundation for developing novel anti-inflammatory drugs that inhibit IL-1? and IL-18 production without interfering with antimicrobial immunity.